Isolation of mouse embryonic fibroblasts (MEFs) from mouse embryos at E12.5

Transfer the uterus containing the embryos to a dish with ice cold PBS.

Separate the embryos by cutting through the uterus in the region between each embryo with scissors.

Transfer each embryo to a new dish with ice cold PBS.

Remove the placenta and other maternal tissues by gently pulling them off using forceps. If the embryo is still in the yolk sac, gently pull the sac off with forceps.

Note: Clean scissors and forceps with 70% ethanol between each embryo.

Pick up the embryo with forceps and transfer to a fresh dish.

Note: Perform steps 6-10 as quickly as possible to avoid the embryo drying out.

Cut off the embryo head (eyes and above).

Note: The head can be used for DNA isolation for genotyping.

Remove the red parts (heart and liver) from the embryo by gently scraping with forceps.

Place the rest of the embryo in a new dish.

Using a scalpel blade, finely chop the embryo into pieces of 1–2 mm.

Note: Use a new scalpel for each embryo.

Add 0.05% trypsin-EDTA (around 7.5 mL), ensuring the embryo pieces are completely covered.

Place the dish in the tissue culture incubator for 10-15 min.

After 10-15 min incubation with 0.05% trypsin-EDTA, check the plate under the microscope. If a lot of cells are floating, proceed to step 13, otherwise incubate for further 5-10 min at 37°C.

Add 7.5 ml MEF culture medium to inactivate the trypsin.

Gently pipet up and down several times.

Transfer the cell suspension to a 15 ml conical centrifuge tube.

Spin tube at 180 x g for 5 min.

Carefully aspirate the supernatant without disturbing the pellet.

Re-suspend the cell pellet in 10 ml of media by gently pipetting up and down several times.

Transfer the cell suspension to a fresh dish.

Transfer the dishes to the incubator.

The following day, replace the old medium with fresh culture medium to get rid of dead cells and debris.

Split the cells when they reach confluency (this is passage number 1). Alternatively, cells can be frozen down (freezing medium: 90% v/v MEF culture medium, 10% v/v DMSO).

MEF obtained by this procedure can be immortalised either by serial passaging, or by SV40-mediated immortalisation.